American Association for Cancer Research
15357163mct130310-sup-fig1-7.pdf (903.91 kB)

Supplementary Figures 1 - 7 from MTI-101 (Cyclized HYD1) Binds a CD44 Containing Complex and Induces Necrotic Cell Death in Multiple Myeloma

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journal contribution
posted on 2023-04-03, 13:42 authored by Anthony W. Gebhard, Priyesh Jain, Rajesh R. Nair, Michael F. Emmons, Raul F. Argilagos, John M. Koomen, Mark L. McLaughlin, Lori A. Hazlehurst

PDF file - 903K, Supplementary Figure S1: MTI-101 is more potent in U266 cell line than linear parent peptide and biotinylated peptides still retain bioactivity; Supplementary Figure S2: Biotin-HYD1 binding complex in NCI-H929 and U266 MM cell lines; Supplementary Figure S3: Overexpression of CD44s in 8226 via retroviral infection results in functional CD44; Supplementary Figure S4: Reduction in syndecan-1 expression failed to confer resistance to MTI-101 induced cell death; Supplementary Figure S5: Reduction in basigin expression failed to confer resistance to MTI-101 induced cell death; Supplementary Figure S6: MTI-101 necrotic cell death in Rip1/Rip3 and Drp1 independent in NCI-H929 cell line, but partially involved in U266; Supplementary S7: MTI-101 has activity as a single agent in 5TGM1 in vivo mouse model.



Our laboratory recently reported that treatment with the d-amino acid containing peptide HYD1 induces necrotic cell death in multiple myeloma cell lines. Because of the intriguing biological activity and promising in vivo activity of HYD1, we pursued strategies for increasing the therapeutic efficacy of the linear peptide. These efforts led to a cyclized peptidomimetic, MTI-101, with increased in vitro activity and robust in vivo activity as a single agent using two myeloma models that consider the bone marrow microenvironment. MTI-101 treatment similar to HYD1 induced reactive oxygen species, depleted ATP levels, and failed to activate caspase-3. Moreover, MTI-101 is cross-resistant in H929 cells selected for acquired resistance to HYD1. Here, we pursued an unbiased chemical biology approach using biotinylated peptide affinity purification and liquid chromatography/tandem mass spectrometry analysis to identify binding partners of MTI-101. Using this approach, CD44 was identified as a predominant binding partner. Reducing the expression of CD44 was sufficient to induce cell death in multiple myeloma cell lines, indicating that multiple myeloma cells require CD44 expression for survival. Ectopic expression of CD44s correlated with increased binding of the FAM-conjugated peptide. However, ectopic expression of CD44s was not sufficient to increase the sensitivity to MTI-101–induced cell death. Mechanistically, we show that MTI-101–induced cell death occurs via a Rip1-, Rip3-, or Drp1-dependent and -independent pathway. Finally, we show that MTI-101 has robust activity as a single agent in the SCID-Hu bone implant and 5TGM1 in vivo model of multiple myeloma. Mol Cancer Ther; 12(11); 2446–58. ©2013 AACR.